1. Field of the Invention
The present invention relates to the derivatives of human C-peptide and its fragments (short-chain analogues) useful for the determination of proinsulin C-peptide level in human serum.
In the radioimmunoassay performed for evaluating the function of pacreatic .beta.-cell, it is an accepted and prevalent notion that the measurement of C-peptide in serum is more reliable in a practical point of view than that of insulin itself in serum, and a number of investigations in connection with this notion have hithertofore been reported (See, for instance, Kaneko; Taisha 10, 1288 (1973), and Saishin-igaku 31, 839 (1976)).
The ground of this notion appeared in these papers may be summarized as follows:
(1) It is difficult to measure accurately the concentration of insulin itself in serum of diabetics who have been or are being treated with insulin, because they hold a naturally occurring antibody against insulin.
(ii) The half-life of insulin in human vascular (vein) is limited to as short as 3-4 minutes whereas that of C-peptide is as long as about 13 minutes, and
(iii) C-Peptide is a peptide which connects the A and B chains of insulin together to constitute the proinsulin molecule and is to be liberated along with an equimolar amount of insulin simultaneously in accordance with the decomposition of proinsulin in pancreatic .beta.-cells.
C-peptide herein referred to is a peptide composed of 31 amino acid residues which correspond to positions 33-63 of human proinsulin, but it has no tyrosine residue. Therefore, C-peptide can not be labeled, as it stands, with radioactive iodine for performing radioimmunoassay.
2. Description of the Prior Art
Steiner and others have therefore proposed the use of an N-tyrosylated C-peptide, the tyrosine residue of which may be labeled with radioactive iodine (Steiner, D. F. et. al.: Proc. Natl. Acad. Sci. U.S., 57, 473 (1967)).
Steiner's tyrosylated C-peptide is regarded as being prepared according to the method described by Melani et. al. (Melani, F. et. al., Proc. Natl. Acad. Sci. U.S., 67, 148 (1970)).
According to this method, the C-peptide may be tyrosylated at pH 7.6 with the N-carboxy anhydride of tyrosine, that is: ##STR1## In this method, however, it is unable to stop the reaction at a predetermined step and, therefore, the concommitant formation of C-peptide derivatives having more than one tyrosines is inevitable.
That is, the product thus obtained is not a single substance but a mixture of C-peptide derivatives, in which one or more tyrosine residues are introduced. The ratios of the components may vary from one lot to another and their separation would be virtually impossible.
Although Steiner contends that the biological activity such as antibody specificity might scarcely be affected by the poly-tyrosylation, it is difficult to affirm his contention in every and all respects.
In general, the introduction of tyrosine residue into a certain peptide may be carried out with one of the following compounds as acylating agent: EQU Z-Tyr(Z)-X (i) EQU Z-Tyr(Bu.sup.t)-X (ii) EQU Z-Tyr-X (iii)
wherein Z represents the benzyloxycarbonyl group, X represents an active group such as Oxysuccinimidyloxy, trichlorophenoxy or azide group and Bu.sup.t represents the tertiary butyl group, and wherein Z may be replaced by Boc (tertiary butoxycarbonyl group) and Bu.sup.t may be replaced by Bzl (benzyl group) in either case. The following paragraphs will describe the use of these compounds for tyrosylation of C-peptide.
A method which employs the agent (i) is disclosed in the specification of Japanese unexamined Pat. No. 25,767/77 and one which utilizes the agent (ii) is reported by Naithani and others (Naithani, V. K., et. al., Hoppe Seyler's Z. Physiol. Chem., 356, 1305 (1975)). In these cases, however, the protecting group on the side chain hydroxyl group of tyrosine has to be removed prior to the labeling.
Thus, the acylation product is subjected to catalytic hydrogenolysis or acidolysis with hydrogen fluoride and the like in the former case, and to acidolysis with trifluoroacetic acid (TFA) (for removal of Bu.sup.t) or hydrogen fluoride (for removal of both Bu.sup.t and Z) in the latter case.
The catalytic hydrogenolysis may be applied to a short chain peptide but hardly applicable to a long chain peptide, because of its low reaction rate. (See: for instance, V. K. Naithani et. al. ibid).
If the acidolytic deprotection is employed, the resultant tyrosine residue tends to undergo modification by the possible action of liberated carbonium cation. Furthermore one can not exclude the possibility that the other part of the C-peptide molecule would undergo modification simultaneously upon acid treatment.
In such cases as those mentioned above, the purification of the product by means of fractionation or the like would present considerable difficulties.
By employing the agent (iii) the above mentioned drawback may be avoided, but the acylation of C-peptide must be done with an excess amount of the reagent to complete reaction. As a result, possible overacylation at the side chain of tyrosine may not be avoided.
The only case in which the overacylation does not occur is when X=N.sub.3 in (iii). However, the tyrosine azide generally has tendency to form an inactive amide in the presence of water during the course of its preparation.
Under the circumstances, the specifications of Japanese Pat. No. 10,872/77 and Japanese Unexamined Pat. No. have proposed the exploitation of a newly defined "C-peptide" which is unique in a sense that it is a derivative of the C-peptide defined by Steiner (a peptide with a sequence of 31 amino acid residues corresponding to positions 33-63 of human proinsulin) and embodies extra sequences Arg-Arg (arginyl-arginine) and Lys(For)-Arg-OH (N.sup..epsilon. -formyllsylarginine) at the N-- and C--terminal positions, respectively, of C-peptide.
This hexatriacontapeptide contains amino acid residues corresponding to positions 31-65 of human proinsulin and has a tyrosine residue at the N-terminal. The hydroxyphenyl group of tyrosine may be labeled with .sup.125 I.
They used this hexatriacontapeptide as antigen in place of C-peptide itself, because the latter had been reported to be only weakly antigenic even in the form of an albumin bound conjugate.
However, the proposed method has a disadvantage in the synthesis of antigen, as it requires operation for coupling 35 amino acid residues as compared to 31 of the normal human C-peptide. The extention of the C-peptide sequence by the four additional residues is deemed to be not necessarily essential for the introduction of the tyrosine residue.